This application claims the benefit of U.S. Provisional Application No. 60/008,375 filed Dec. 8, 1995 and U.S. Provisional Application No. 60/020,163 filed Jun. 20, 1996.
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and gene theapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with modified fibers for the purpose of cell-specific targeting.
2. Description of the Related Art
Recombinant adenoviruses have demonstrated great utility in the context of a variety of strategies to accomplish gene therapy (1-3). One of the principal features of recombinant adenoviruses resulting in their frequent use relates to the unique ability of these vectors to accomplish direct in vivo gene delivery. In this regard, recombinant adenoviral vectors have been shown to be capable of efficient gene transfer to parenchymal cells of various organs including the lung, the brain, the pancreas, the gall bladder, the liver, and others (4-12). This has allowed the utilization of recombinant adenoviral vectors as an approach to treat inherited genetic diseases, such as cystic fibrosis, whereby the delivered vector may be contained within the target organ (4-13). In addition, the ability of the adenoviral vector to accomplish in situ tumor transduction has allowed the development of a variety of anti-cancer gene therapy approaches for loco-regional disease (14-18, 45). Again, these approaches have been directed towards non-disseminated disease, whereby vector containment favors tumor cell-specific transduction.
Despite the versatility of the adenoviral vector in these contexts, the full utility of the recombinant virions for in vivo gene transfer applications is not currently exploitable. This is because the promiscuous tropism of the virus allows widespread, unrestricted tissue transduction after systemic in vivo vector delivery (19, 20). Thus, approaches based upon vascular vector delivery to specific organ sites would be undermined by ectopic, non-targeted cellular transduction. This biologic feature of the virion has thus limited gene therapy approaches to the aforementioned loco-regional or compartment disease models whereby anatomic containment favors some level of selective target cell transduction.
Adenoviruses as vectors for gene therapy
The approach of direct intramuscular injection, whether of naked DNA or of viral vectors, as a method for in vivo gene transfer in various genetic diseases suffers from practical limitations. The injection of the large mass of skeletal tissue would be impractical in a clinical context. However, the problems associated with Intramuscular injection could be avoided by the targeted delivery to muscle cells of an intravenously administered vector. Adenoviral vectors can accomplish in vivo gene delivery to a variety of organs after intravenous injection. In these instances, gene transfer frequencies have been sufficiently high to correct inherited metabolic abnormalities in various murine models. Thus, adenoviral vectors fulfill two requirements of an intravenously administered vector for gene therapy: systemic stability and the ability to accomplish long-term gene expression following high efficiency transduction of muscle cells. However, adenoviruses suffer from the disadvantage that the widespread distribution of the adenovirus cellular receptor precludes the targeting of specific cell types. This lack of tropism of adenoviral vectors would result in a decrease in the efficiency of transduction, as the number of virus particles available for delivery to the target cells would be decreased by sequestration by nontarget cells. Furthermore, this would allow ectopic expression of the delivered gene, with unknown and possibly deleterious consequences. Therefore a means must be developed to redirect the tropism of the adenovirus vector specifically to target cells to permit gene delivery uniquely to organs affected.
Another recognized problem with the use of existing adenovirus vectors deleted only in the E1 region of the genome is that the low-level expression of late adenovirus gene products triggers an immune response in the host. This is manifested as an inflammatory immune attack on the transduced cells which leads to transient expression of the transgene and precludes repeated gene transfer with the same vector. A further problem associated with the current generation of adenovirus vectors is that the insert capacity is presently limited to about 7.5 kb, whereas many genes cDNA's are much greater in length.
A strategy to overcome this limitation would be the modification of the cell binding domains of the adenovirus to allow interaction with cellular receptors in a specific manner. Adenovirus interacts with eucaryotic cells by virtue of specific receptor recognition by domains in the knob portion of the fiber protein (21-23) which protrude from each of the twelve vertices of the icosahedral capsid.
The prior art is deficient in the lack of effective means to produce recombinant adenoviral vectors with modified fibers for purposes of cell-specific targeting. The present invention fulfills this longstanding need and desire in the art.